Project Summary/Abstract t The number of Americans over age 65 is 48 million in 2015, and it is expected to be 98million in 2060 (U.S. Census). People over age 65 typically suffer from increased white adipose tissue (WAT) accumulation, especially in the visceral (abdominal) area. Visceral adiposity accelerates aging by promoting insulin resistance, cardiovascular dysfunction, and many other chronic health conditions, significantly shortening healthspan and lifespan. Thus, preventing or reducing adipose tissue accumulation is critical for healthy aging. Unfortunately, the relationship between aging and adipose tissue accumulation is poorly understood. Adipocytes (fat cells) undergo hypertrophy (cell enlargement) during aging, but it remains unclear if adipose tissue also expands through adipogenesis (the generation of new adipocytes), which will grant adipose tissue with unlimited potential to grow. The rate of adipogenesis is very low in young mice, similar to that in young humans. Preliminary data of this proposal show the first cellular evidence that WAT expands with age through massive adipogenesis. Thus, in contrast to most adult stem cells that exhibit a reduced ability to proliferate and differentiate, adipogenesis of preadipocytes is unlocked by aging. The goal of this R01 proposal is to determine when, where and why does adipogenesis takes place during aging. Based on the preliminary findings, the hypothesis is that aging generates new preadipocytes which undergo adipogenesis to become new adipocytes, and the stromal microenvironment generated by cellular senescence plays a key role in this process. Proposed experiments in Aim 1 will use AdipoChaser mice to determine the exact age stage when adipogenesis takes place, and use MuralChaser mice to determine the percentage of new preadipocytes generated during aging and their rate of adipogenesis. Unbiased single-cell sequencing will also be used to identify preadipocyte subpopulations newly generated during aging. Proposed experiments in Aim 2 will use both in vitro differentiation assays and in vivo transplantations to determine the contribution of paracrine stimulation to age-associated adipogenesis. Proposed experiments in Aim 3 will determine whether eliminating senescent cells prevents age-associated adipogenesis, and assess whether the adipogenesis-inhibitory cells (CD142+) have declined function during aging (through senescence) and thus contribute to the unlocked adipogenesis. The proposed research provides novel scientific knowledge that adipogenesis is the major contributor to age-associated adiposity, and mechanistic insights into why adipogenesis is unlocked by aging. Successful completion of the proposed research will provide new therapeutic avenues for the prevention and treatment of age-associated adiposity and its related chronic diseases, ultimately promoting longevity and healthy aging.